We have constructed and identified cDNA clones of human parainfluenza type 3 virus (PIV3) mRNAs encoding the hemagglutinin-neuraminidase (HN) glycoprotein, fusion (F) glycoprotein, major nucleocapside protein (NP), nucleocapsid phosphoprotein (P), and matrix (M) protein. The P mRNA contains a second, overlapping open reading frame that encodes the nonstructural (C) protein. Complete nucleotide sequences have been determined for the HN, F, NP, P+C and M mRNAs. Synthetic oligonucleotides were used to direct dideoxynucleotide sequencing of gene junctions in PIV3 genomic RNA (UpsilonRNA). From the sequencing of UpsilonRNA, a seventh viral gene was detected and was identified as the L gene by RNA blot hybridization. The order of the six PIV3 genes on UpsilonRNA is 3'-NP-P+C-M-F-HN-L. The five intergenic regions consist of the trinucleotide 3'-GAA. These studies show that PIV3 encodes six mRNAs (NP, P+C, M, F, HN and L) that encode seven proteins (NP, P, C, M, F, HN, and L). Preliminary studies on the expression of the F and HN genes by recombinant vaccinia viruses and SV40 viruses are described, together with the construction of new SV40 vectors and a new strategy for rapid mutagenesis and expression.